Murine studies have demonstrated that liver grafts can survive permanently when transplanted into hosts that have not been immunologically suppressed (Qian, et al., Hepatology, 19:916 (1994)). Similarly, these transplanted liver grafts have also been shown to induce donor-specific acceptance of skin and cardiac grafts (Qian, et al., Hepatology, 19:916 (1994)). The inventors believe that such graft acceptance in a host depends on the presence of bone marrow-derived leukocytes in the transplanted specimen (Starzl, et al., Lancet, 339:1579 (1992); Starzl, et al., Immunol. Today, 14:326 (1993); Starzl, et al., Hepatology, 17:1127 (1993)).
Following transplantation, the migration of bone-marrow derived cells from whole organs appears to result in a low level chimerism and donor-specific immunological non-reactivity (Starzl, et al., Lancet, 339:1579 (1992); Starzl, et al., Immunol. Today, 14:326 (1993); Qian, et al., Hepatology, 19:916 (1994)). The inventors believe that cell migration and chimerism may be the basis for acceptance of all allografts (Starzl, et al., Lancet, 339:1579 (1992); Starzl, et al., Immunol. Today, 14:326 (1993); Starzl, et al., Hepatology, 17:1127 (1993)). Thus, the exceptional tolerogenicity of the liver could mirror a comparatively heavy content of immature or progenitor DC within this organ (Steinman, et al., Hepatology, 17:1153 (1993)).
Additional studies by the inventors have shown that the propagation of myeloid lineage cells from normal mouse heart non-parenchymal cells (NPC) in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) is difficult to achieve. The possibility that multiple hematolymphopoietic lineages participate in the leukocyte traffic occurring after an organ transplant has been suggested from observations in rats (Demetris, et al., Transplant. Proc., 25:3337 (1993)) and humans (Starzl, et al., Hepatology, 17:1127 (1993)), and demonstrated unequivocally in mouse liver transplant experiments (Qian, et al., Hepatology, 19:916 (1994)). These lineages may survive permanently without treatment in mice (Qian, et al., Hepatology, 19:916 (1994)) or subsequent to an immunosuppressive regimen in rats (Demetris, et al., Transplant. Proc., 25:3337 (1993)) and, in some cases, humans (Starzl, et al., Hepatology, 17:1127 (1993)). The probability of pluripotent stem cells and immature DC--amongst other lineages--in the bone marrow-derived interstitial population of non-lymphoid organs is inherent in the inventors' cell migration and chimerism explanation (Starzl, et al., Lancet, 339:1579 (1992); Starzl, et al., Immunol. Today, 14:326 (1993)).
Once a graft specimen has been transplanted, the bone marrow-derived leukocytes, otherwise known as "passenger leukocytes," migrate from the donor specimen into the host's tissue (Starzl, et al., Lancet, 339:1579 (1993); Starzl, et al., Immunol. Today, 14:326 (1993)). Once in the host's tissue, evidence exists that a complex of interactions between the donor's leukocytes and the host's immune system can lead to a decreased immune response in the host, and even to tolerance induction in certain situations (Starzl, et al., Lancet, 339:1579 (1993); Starzl, et al., Immunol. Today, 14:326 (1993); Demetris, et al., Transplant. Proc., 25:3337 (1993); Starzl, et al., Hepatology, 17:1127 (1993); Yoshimura, et al., Transplantation, 49:167 (1990); Thomas, et al., Transplantation, 51:198 (1991); Van Twuyver, et al., N. Engl. J. Med., 325:1210 (1991); Barber, et al., Transplantation, 51:70 (1991)). Numerous reports also exist indicating that mature T-cells can be tolerized to allogeneic antigens outside the thymus (Murphy, et al., Proc. Natl. Acad. Sci. U.S.A., 86:10034 (1989); Miller, et al., Annu. Rev. Immunol., 10:51 (1992)). Accordingly, chimeric cells present in the periphery may play a key role in achieving allotolerance.
HLA-DR.sup.dim allogeneic donor bone marrow cells showing veto cell activity--inactivation of T-helper cells or cytotoxic T-cell precursors--have also been postulated to be immature DC (Thomas, et al., Transplantation, 57:101 (1994)). These immature DC were shown by the present inventors to have avid phagocytic activity in culture and might be expected, therefore, to elicit a deviant, or tolerogenic, local and systemic immune response shortly after injection thereof. The precise basis of the DC-T-cell interaction leading to tolerance induction is not known at this time. Nonetheless, such an interaction would logically depend on the relative affinity or avidity of the donor DC-T-cell receptors ("TCR"), and on the expression of adhesions and co-stimulatory molecules (e.g., B7-1 and B7-2) on the former cells.
An example of a thoroughly studied atypical or "deviant" cytokine-modulated immune response induced by bone marrow-derived antigen-presenting cells ("APC") is provided by Streilein et al. (Streilein, et al., J. Neuroimmunol., 39:185 (1992); Wilbanks, et al., J. Immunol., 146:2610 (1991)). Streilein and his colleagues studied class II.sup.dim APC with dendritic morphology, which are now believed to be variant DC in the iris, ciliary body, and other tissues lining the anterior chamber of the eye (Wilbanks, et al., J. Immunol., 146:3018 (1991); Streilein, et al., J. Neuroimmunol., 39:185 (1992)). After APC took up bovine serum albumin ("BSA") injected into the anterior chamber of test specimens, the BSA was presented ineffectively to local T-cells, and subsequently in the spleen when APC-peptide complexes arrived there. As a consequence, the test specimen experienced both a dampened systemic and local, or ocular, immune response when challenged with antigen. Although DC precursors appear to be concentrated in the liver, similar subpopulations presumably exist in all other tissues and whole organs, and particularly in the bone marrow, which constitutes a major source of leukocytes.
The liver, which is the most tolerogenic whole organ (Starzl, et al., Hepatology, 17:1127 (1993); Starzl, et al., Surgery, 58:131 (1965);
Calne, et al., Nature, 223:472 (1969)), possesses a relatively large concentration of DC. And across most mouse strain combinations, a liver can be transplanted without host immunosuppression (Qian, et al., Hepatology, 19:916 (1994)). Although interstitial liver DC have been characterized by immunohistochemical studies in both rodents (Hart, et al., J. Exp. Med., 154:347 (1981); Steiniger, et al., Transplantation, 38:169 (1984)) and humans (Prickett, et al., Transplantation, 46:754 (1988)), there is a dearth of published data concerning the in vitro properties of liver DC, and there have been no known attempts to propagate DC from a normal liver.
Reports indicate that the destination of the passenger leukocytes after whole organ transplant is lineage-specific, following the same routes taken by syngeneic cells of the same lineages (Demetris, et al., Transplant. Proc., 25:3337 (1993); Qian, et al., Hepatology, 19:916 (1994)). Moreover, the bone marrow-derived DC lineage is postulated to be the most important of the passenger leukocytes, providing variable degrees of donor-specific non-reactivity (Starzl, et al., Lancet, 339:1579 (1993); Starzl, et al., Immunol. Today, 14:326 (1993); Demetris, et al., Transplant. Proc., 25:3337 (1993)).
Although it has previously been postulated that complex interactions between donor leukocytes and a host's immune system may lead to a decreased immune response by the host (Starzl, et al., Lancet, 339:1579 (1993); Starzl, et al., Immunol. Today, 14:326 (1993); Demetris, et al., Transplant. Proc., 25:3337 (1993); Starzl, et al., Hepatology, 17:1127 (1993); Yoshimura, et al., Transplantation, 49:167 (1990); Thomas, et al., Transplantation, 51:198 (1991); Van Twuyver, et al., N. Engl. J. Med., 325:1210 (1991); Barber, et al., Transplantation, 51:70 (1991)), and even to tolerance induction, reports of attempts to propagate DC lineage cells from a normal liver are not known to exist.
And despite the fact that DC progeny from mouse blood and bone marrow have been propagated and matured in the presence of GM-CSF (Inaba, et al., J. Exp. Med., 175:1157 (1992); Inaba, et al., J. Exp. Med., 176:1693 (1992)), attempts by the inventors to culture liver-derived DC in the presence of a cytokine alone, such as GM-CSF, failed to achieve appreciable differentiation of liver DC in vitro. The additional presence of tumor necrosis factor-.alpha. ("TNF-.alpha.") and/or interferon-.gamma. ("INF-.gamma.") was likewise found by the inventors to be ineffective in raising the level of major histocompatibility complex ("MHC") class II expression in mouse-derived liver DC. Prior to the present invention, no known means existed for maturing DC derived from an hepatic source.
The present invention provides a method of propagating immature or undifferentiated mammalian DC in the presence of a cytokine, which may then be used to induce donor-specific tolerance to grafts transplanted to a host mammal. The present invention further provides a method of producing immunologically mature mammalian DC in the presence of a cytokine and an extracellular matrix protein, which may then be used to enhance the immune response, including antibody production and cell-mediated immunity, of a host mammal.